Biochemical characterization of benzaldehyde dehydrogenases from petunia

Benzoic acid, the simplest aromatic carboxylic acid, is an important building block for a wide range of primary and specialized plant metabolites. In Petunia hybrida, benzoic acid serves as a key precursor of volatile benzenoids, which are responsible for the primary floral scent. However, the enzymes responsible for benzoic acid production in plants have rarely been reported. This study aimed to identify and characterize benzaldehyde dehydrogenases-enzymes that catalyze the oxidation of benzaldehyde to benzoic acid-using a combination of metabolite analysis and transcriptomic approaches. We identified two petunia benzaldehyde dehydrogenases, PhBALDH-1 and PhBALDH-2, with apparent K_m values of 93 and 51 μM for benzaldehyde, respectively. While PhBALDH-2 exhibited a strong preference for NAD⁺ as a cofactor, PhBALDH-1 was capable of utilizing both NAD⁺ and NADP⁺. In vitro mutagenesis experiments demonstrated that substituting a single amino acid markedly affected the cofactor specificity of the PhBALDH-1 enzyme. Gene expression analysis during petunia flower development suggests that both PhBALDH-1 and PhBALDH-2 are likely involved in regulating volatile benzenoid biosynthesis in petunia flowers. Our findings provide functional insights into the biosynthesis of benzoic acid and its regulation in P. hybrida.